DESCRIPTION: A unique and integral component of the bacteria cell wall of gram-negative bacteria is the lipopolysaccharide (LPS) layer. The LPS layer has been shown to be important in the development of virulent strains of bacteria and thus selective interference with LPS formation would provide a selective strategy for the design of novel antibiotics. A key enzyme in the biosynthetic pathway for the formation of lipopolysaccharides is 2-keto-3-deoxy-D-manno-2-octulosonic acid-8-phosphate (KDO8P) synthase. The studies described in this proposal are directed toward providing an in-depth understanding of the catalytic mechanism of KDO8P with the ultimate goal of impacting inhibitor design. The current proposal is based upon a transient kinetic approach using rapid chemical quench and stopped-flow fluorescence methodologies. The important advantage of this approach is the ability to observe directly events occurring at the active site including binding events, transient enzyme intermediates, protein conformational changes, and catalysis. The rate constants of individual steps can be measured directly and any enzyme intermediates including different conformational species which might be formed can be observed directly. The specific aims are: (1) Provide a complete kinetic and thermodynamic description of the KDO8P reaction pathway. (2) Isolation and characterization of enzyme intermediates. (3) Probe the catalytic mechanism through the use of alternate analogs of PEP. (4) Site-directed mutagenesis to identify amino acids residues important for recognition and catalysis.